Extracellular matrix (ECM) remodeling is a process that is crucial to the development of embryos, the growth and metastasis of tumors, and wound healing and homeostasis of tissues in adults. As such, it involves dozens of gene products that are regulated by mechanisms operating at transcriptional and multiple posttranslational levels. This complexity of regulation has made the development of a comprehensive understanding of the biology of ECM remodeling in vivo an unusually challenging task, yet such an understanding would be of profound value to our knowledge of and clinical approaches to the treatment of many cancers. The primary effectors of ECM remodeling are the matrix metalloproteinases (MMPs). Homologs of this gene family have been identified in every metazoan examined. We propose that the zebrafish embryo is an ideal system for the study of the regulation of MMP activity, and we present some progress we have made in the development of this organism as a platform for MMP research. We have identified 25 genes encoding MMPs in the zebrafish genome, and 5 genes encoding their endogenous inhibitors, the tissue inhibitors of MMPs. Based on a phylogenetic analysis, we have identified the most probable homologies of these sequences and found that there are two that are of equivocal identity. We have developed 17 antibodies specific to zebrafish MMPs and have begun characterizing the ontogeny of these molecules. Finally, we have developed two novel assays that allow the detection and characterization of active MMPs in vivo (differential in vivo zymography and activity-based protease profiling). In combination with the array of powerful biochemical, genomic, cell, and molecular biological techniques available to zebrafish researchers already, we feel that these new reagents and techniques make the zebrafish the best model system for the study of MMP regulation currently available.
ABSTRACT:Objectives: We sought to determine whether rotenone, a commonly used pesticide, exhibits neurotoxicity in zebrafish by causing dopamine neuron loss through rotenone-induced oxidative damage. Methods: We exposed transgenic zebrafish embryos expressing green fluorescent protein under the control of the cis-regulatory elements of dopamine transporter (dat) to rotenone to determine the neurotoxic effects of rotenone on dopamine neuron abundance and pattern distribution, as well as the presence of apoptotic markers. The oxidative stress potential of rotenone on embryos was assessed using a live MitoSOX Red assay, and behavioural testing on adult zebrafish was assessed using video recordings of midline crossing events. Results: Zebrafish embryos treated with rotenone displayed a 50% reduction in dopamine neurons in the ventral diencephalon when exposed to 30µM rotenone (n=6, p<0.001), and rotenone-exposed zebrafish raised to adulthood demonstrate an anxiety-like behaviour (n=5, p<0.01). Furthermore embryos exposed to rotenone also demonstrated a logarithmic increase in markers of oxidative damage (n=3, p<0.001) and apoptotic activity in their diencephalic neurons. Conclusions: These results show that rotenone can induce dopamine neuron loss in zebrafish, providing a useful model for studying the environmental causes of Parkinson’s disease. RÉSUMÉ:Objectif: Nous cherchons à déterminer si la roténone, un élément commun dans les pesticides, démontre de la neurotoxicité dans les poissons-zèbres en causant une perte de dopamine dans leurs neurones à travers le dommage oxydatif induit par la roténone. Méthode: Nous avons exposé des embryons de poissons-zèbres transgéniques qui expriment la protéine fluorescente verte sous le contrôle d’éléments cis-régulateurs des transporteurs sélectifs de dopamine (dat) à la roténone pour déterminer les effets neurotoxiques de ce dernier sur les niveaux dopaminergiques dans leurs neurones. De plus, nous avons évalué la présence de marqueurs apoptotiques. Le stress oxydatif potentiel de la roténone sur les embryons a été analysé par le « live MitoSOX Red assay » et les tests comportementaux sur les poissons-zèbres adultes furent analysés en utilisant des enregistrements vidéo. Résultats: Les embryons de poissons-zèbres qui ont été traités avec la roténone ont démontré une réduction de dopamine de 50% dans les neurones localisés dans le diencéphale ventral, quand exposés à 30µM de roténone (n=6, p<0.001). Ils ont également illustré une augmentation logarithmique dans les marqueurs de dommage oxydatif (n=3, p<0.001) et une activité apoptotique dans les neurones du diencéphale. Les poissons-zèbres exposés à de la roténone qui ont atteint l’âge adulte ont démontré des comportements d’anxiété (n=5, p<0.01). Conclusion: Les résultats démontrent que la roténone peut induire une perte dopaminergique dans les neurones des poissons-zèbres. Ces résultats s’avèrent utiles pour étudier davantage les causes environnementales reliées à la maladie de Parkinson.
Extracellular matrix (ECM) remodeling is a physiologically and developmentally essential process mediated by a family of zinc-dependent extracellular proteases called matrix metalloproteinases (MMPs). In addition to complex transcriptional control, MMPs are subject to extensive post-translational regulation. Because of this, classical biochemical, molecular and histological techniques that detect the expression of specific gene products provide useful but limited data regarding the biologically relevant activity of MMPs. Using benzophenone-bearing hydroxamate-based probes that interact with the catalytic zinc ion in MMPs, active proteases can be covalently ‘tagged’ by UV cross-linking. This approach has been successfully used to tag MMP-2 in vitro in tissue culture supernatants, and we show here that this probe tags proteins with mobilities consistent with known MMPs and detectable gelatinolytic activity in homogenates of zebrafish embryos. Furthermore, because of the transparency of the zebrafish embryo, UV-photocroslinking can be accomplished in vivo, and rhodamated benzophenone probe is detected in striking spatial patterns consistent with known distributions of active matrix remodeling in embryos. Finally, in metamorphosing Xenopus tadpoles, this probe can be used to biotinylate active MMP-2 by injecting it and cross-linking it in vivo, allowing the protein to be subsequently extracted and biochemically identified.
Purpose: The Clinician Investigator Trainee Association of Canada/ Association des cliniciens-chercheurs en formation du Canada (CITAC/ACCFC) recently published the first survey to assess factors contributing to trainee satisfaction. One key finding is that increased level of mentorship strongly correlates with overall satisfaction; however, while 98% of respondents reported mentorship as important to success, more than 60% expressed some dissatisfaction with the mentorship received. To help address this discrepancy, we reviewed mentorship in academic medicine, focusing on clinician-investigator trainees, and distilled a set of recommendations for mentors, mentees and institutions. Source: OVID and manual curation based on the search terms ‘mentorship’ AND ‘education, medical and research’ identified 198 articles. Two authours independently reviewed both titles and abstracts and narrowed them down to 75 articles, based on relevance to mentorship in academic medicine. Consensus resulted in the selection of 19 articles for detailed review. Principal findings and Conclusion: Mentorship is beneficial at each training stage and is associated with greater research productivity, career retention and promotion. Nevertheless, more rigorous studies are needed, especially regarding cost-effectiveness. Studies have identified the characteristics of good mentors, including the ability to ensure open communication, ability to maintain confidentiality and ability to ensure that there is no mentor-mentee competition. Similarly, the characteristics of good mentees have been identified as the ability to take ownership of a project and the ability to build a network or team of mentors. The literature has also identified the actions that institutions can take to facilitate mentorship, which include mentor training and recognizing mentorship through awards.
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